Method and apparatus for determining red blood cell indices of a blood sample utilizing the intrinsic pigmentation of hemoglobin contained within the red blood cells

ABSTRACT

A method for the determination of the red blood cell indices including the volume, and hemoglobin content and concentration for individual red blood cells, as well as red blood cell population statistics, including total number of red blood cells present in the sample, and mean values for each of the aforementioned indices within a substantially undiluted blood sample is provided.

The present application is entitled to the benefit of and incorporatesby reference essential subject matter disclosed in the following U.S.Provisional Patent Applications: Ser. No. 61/038,557, filed Mar. 21,2008; 61/038,574, filed Mar. 21, 2008; 61/038,545, filed Mar. 21, 2008;and 61/038,559, filed Mar. 21, 2008.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to apparatus and methods for analysis ofblood samples in general, and for the determination of a red bloodcell's volume as well as the mean cell volume of a sample in particular.

2. Background Information

Physicians, veterinarians and scientists have examined human andanimals' biologic fluids, especially blood, in order to determineconstituent particulate quantities as well as to identify the presenceof unusual particulates not seen in healthy subjects. The particulatesgenerally measured, quantified and identified include red blood cells(RBCs), white blood cells (WBCs), and platelets. RBC analyses caninclude determinations of RBC number, size, volume, shape, hemoglobincontent and concentration, and the hematocrit (also referred to as thepacked cell volume). RBC analyses can also involve determining thepresence and/or concentration of certain components within the red cellssuch as DNA, RNA, including the detection of the presence and/orenumeration of hematoparasites (e.g., malarial parasites) either in theRBCs or trypanosomes which are extracellular or leishmaniasis organismswhich are in the WBCs as well as many other hematoparasites. WBCanalyses can include a determination of the population frequency of WBCsub types generally referred to as a differential WBC count, as well asthe notification of any unusual cell types not found in healthysubjects. Platelet (or in certain animals including birds, reptiles andfish, thrombocytes which are similar in function to platelets in mammalsbut are about ten times larger and nucleated) analyses can includeplatelet number, size, shape texture, and volume determinations,including determining the presence of clumps of platelets orthrombocytes within the sample.

Known blood examination techniques, described in detail medical textssuch as Wintrobe's Clinical Hematology 12^(th) Edition, generally dividethe examination methods into manual, centrifugal, and impedance typemethods. Manual methods for cell enumeration typically involve thecreation of an accurately determined volume of a blood or fluid samplethat is quantitatively diluted and visually counted in a countingchamber. Manual examination methods include examining a peripheral smearwhere the relative amounts of the particulate types are determined byvisual inspection. Centrifugal examination methods involve centrifugingthe sample, causing the sample to separate into constituent layersaccording to the relative densities of the constituents. The componentlayers can be stained to enhance visibility or detection. Impedancemethods involve the examination of an accurate volume of blood which istreated according to the particulate being measured; e.g., lysing RBCsfor enumeration of the nucleated cells and volumetrically diluting thesample in a conductive fluid. The process typically involves monitoringa current or voltage applied to sample passing through a narrow passageto determine the effect particles have on the current/voltage as theparticles pass through in single file. Other techniques involveanalyzing the intensity and angle of scatter of light incident toparticulates passing single file through a light beam. Flow cytometricmethods can also be used that involve staining particulates of interestin suspension with fluorophores attached to antibodies directed againstsurface epitopes present on cell or particle types, exciting the stainedparticulates with light of appropriate wavelengths, and analyzing theemission of the individual particulates/cells.

All of the aforementioned methods, other than the peripheral smear orcentrifugal separation, require dispensing a precise volume of sample.Inaccuracies in the sample volume will result in quantitative errors ofthe same magnitude in the associated analysis. With the exception ofcentrifugal methods, all of the aforementioned methods also require thesample to be mixed with one or more liquid reagents or diluents, andalso require calibration of the instrument to obtain accurate results.In the case of peripheral smears, a high degree of training is needed toproperly examine the smear. A number of the aforementioned methodsgenerate large volumes of contaminated waste which is expensive tohandle. Additionally, the above-described methods are not suitable todetermine the complete blood count (CBC) in birds, reptiles and fishwhere the red blood cells and thrombocytes are nucleated and in certainmammals where the red blood cells size is very small and may be confusedwith platelets.

The amount of information that can be determined by examining the bloodof a human or animal is vast. It is particularly useful to determine theindices of RBCs; e.g., individual cell size, individual cell hemoglobincontent and concentration, and population statistics of RBCs within asample. The mean and dispersion statistics (e.g., coefficients ofvariation) for each of the aforementioned parameters can also provideimportant information, as is evidenced by their discussion within theabove-referenced text by Wintrobe, which has enabled physicians tobetter categorize disorders of RBCs.

SUMMARY OF THE INVENTION

According to the present invention, a method for the determination ofthe RBC indices including the volume, and hemoglobin content andconcentration for individual RBCs, as well as RBC population statistics,including total number of RBCs present in the sample, and mean valuesfor each of the aforementioned indices within a substantially undilutedblood sample is provided.

According to an aspect of the present invention, a method is providedincluding the steps of: 1) providing a substantially undiluted bloodsample; 2) depositing the sample into an analysis chamber adapted toquiescently hold the sample for analysis, the chamber defined by aninterior surface of a first panel, and an interior surface of a secondpanel, wherein both panels are transparent, and the chamber has a heightextending between the interior surfaces of the panels, which height issuch that at least one red blood cell within the sample contacts both ofthe interior surfaces; 3) imaging the at least one red blood cellcontacting the interior surfaces; 4) determining an optical density of aportion of the imaged red blood cell contacting both interior surfaces;and 5) determining the hemoglobin concentration of the red blood cellcontacting the interior surfaces, using the determined optical densityvalue of the pixel optically aligned with the portion of the red bloodcell extending between the interior surfaces.

According to another aspect of the present invention, a method fordetermining a cell volume of a red blood cell within a substantiallyundiluted blood sample is provided. The method includes the steps of: 1)providing a substantially undiluted blood sample; 2) depositing thesample into an analysis chamber adapted to quiescently hold the samplefor analysis, the chamber defined by an interior surface of a firstpanel, and an interior surface of a second panel, wherein both panelsare transparent, and the chamber has a height extending between theinterior surfaces of the panels, which height is such that at least onered blood cell within the sample contacts both of the interior surfaces;3) imaging the at least one red blood cell contacting the interiorsurfaces, including a portion of the red blood cell in contact with bothinterior surfaces; 4) determining the average optical density of theportion of the red blood cell in contact with both interior surfaces; 5)determining the total optical density of the entire red blood cell; and6) determining the cell volume of the at least one red blood cell usingthe chamber height, the average optical density determined for theportion of the imaged red blood cell contacting both interior surfaces,and the optical density determined for the entire imaged red blood cell.

An advantage of the present invention is that it can be used todetermine characteristics of a blood sample using an extremely smallsample volume that may be obtained directly from the patient bycapillary puncture rendering it more useful for point of careapplication or from a venous sample if desired.

Another advantage of the present invention is that it is operable todetermine characteristics of a blood sample using the intrinsicpigmentation of hemoglobin, and therefore does not need the addition ofany dyes or stains. The high molar extinction coefficient of hemoglobinpermits accurate determinations of its relative or absoluteconcentration within very small light path distances, as small as a fewmicrons.

Another advantage of the present invention is that it makes it possibleto determine individual RBC indices on a particular cell, so thatassociations between indices may be identified.

Another advantage of the present method is that it operates free ofexternal and internal fluidics, and independent of gravity ororientation, and therefore is adaptable for use in a hand held deviceand in microgravity conditions.

Another advantage of the present method is that unlike impedancecounters the present apparatus need not be calibrated each time it isused. The present apparatus is also not subject to pertebrations such asthe shape of the cells, the orientation of the cells as they flowthrough an impedance type cell counters orifice for measurement, or theosmolality effects of the dilutant fluids needed for impedance counting.

The present method and advantages associated therewith will become morereadily apparent in view of the detailed description provided below,including the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-4 are cross-sectional diagrammatic representations of analysischambers that may be used in the present method.

FIG. 5 is a diagrammatic planar view of a tape having a plurality ofanalysis chambers.

FIG. 6 is a diagrammatic planar view of a disposable container having ananalysis chamber.

FIG. 7 is a diagrammatic cross-sectional view of a disposable containerhaving an analysis chamber.

FIG. 8 is a diagrammatic schematic of an analysis device that may beused with the present method.

FIG. 9 is an enlarged view of a portion of the analysis chamber shown inFIG. 1.

FIG. 10 is a block diagram illustrating method steps for determining thehemoglobin concentration within a red blood cell, and the meanhemoglobin concentration within a plurality of red blood cells accordingto an aspect of the present invention.

FIG. 11 is a block diagram illustrating method steps for determining thecell volume of a red blood cell, and the mean cell volume of apopulation of red blood cells according to an aspect of the presentinvention.

FIG. 12 is a block diagram illustrating method steps for determining thehemoglobin content of a red blood cell, and the mean hemoglobin contentof a population of red blood cells according to an aspect of the presentinvention.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present method and apparatus for analyzing a sample of substantiallyundiluted whole blood allows the determination of the red blood cell(RBC) cell volume (CV), mean cell volume (MCV), cell hemoglobinconcentration (CHC), mean cell hemoglobin concentration (MCHC), and themean cell hemoglobin content (MCH), as well as their populationstatistics, without the addition of any dyes, reagents (other thananticoagulants in some embodiments) or diluents to the sample.

The present method utilizes an analysis chamber that is operable toquiescently hold a sample of substantially undiluted anticoagulatedwhole blood for analysis. The chamber is typically sized to hold about0.2 to 1.0 μl of sample, but the chamber is not limited to anyparticular volume capacity, and the capacity can vary to suit theanalysis application. The phrase “substantially undiluted” as usedherein describes a blood sample which is either not diluted at all orhas not been diluted purposefully, but has had some reagents addedthereto for purposes of the analysis. To the extent the addition of thereagents dilutes the sample, if at all, such dilution has no clinicallysignificant impact on the analysis performed. Typically, the onlyreagents that will be used in performing the present method areanticoagulants (e.g., EDTA, heparin) and in some instances anisovolumetric sphering agent. These reagents are generally added indried form and are not intended to dilute the sample. Under certaincircumstances (e.g., very rapid analysis), it may not be necessary toadd the anticoagulating agent, but it is preferable to do so in mostcases to ensure the sample is in a form acceptable for analysis. Theterm “quiescent” is used to describe that the sample is deposited withinthe chamber for analysis, and the sample is not purposefully movedrelative to the chamber during the analysis; i.e., the sample residesquiescently within the chamber. To the extent that motion is presentwithin the blood sample, it will predominantly be that due to Brownianmotion of the blood sample's formed constituents, which motion is notdisabling of the use of the device of this invention.

Now referring to FIG. 1, the analysis chamber 10 is defined by a firstpanel 12 having an interior surface 14, and a second panel 16 having aninterior surface 18. The panels 12, 16 are both sufficiently transparentto allow the transmission of light along predetermined wavelengths therethrough in an amount sufficient to perform the optical density analysisdescribed below. At least a portion of the panels 12, 16 are parallelwith one another, and within that portion the interior surfaces 14, 18are separated from one another by a height 20 such that at least someindividual RBCs 22 within a sample each individually contact bothinterior surfaces 14, 18, and/or one or more aggregates 23 of RBCswithin the sample each contact both interior surfaces 14, 18 of thechamber panels 12, 16, and one or more RBC void areas (e.g., lacunae) 24within the quiescent sample extend between the interior surfaces, aswill be discussed in greater detail below. The present method canutilize a variety of different analysis chambers types having theaforesaid characteristics, and is not therefore limited to anyparticular type of analysis chamber. An analysis chamber having parallelpanels 12, 16 simplifies the analysis and is therefore preferred, but isnot required for the present invention; e.g., a chamber having one paneldisposed at a known non-parallel angle relative to the other panel couldbe used.

Now referring to FIGS. 2-5, an example of an acceptable chamber 10 isshown that includes a first panel 12, a second panel 16, and at leastthree separators 26 disposed between the panels 12, 16. The separators26 can be any structure that is disposable between the panels 12, 16,operable to space the panels 12, 16 apart from one another. Thedimension 28 of a separator 26 that extends between the panels 12, 16 isreferred to herein as the height 28 of the separator 26. The heights 28of the separators 26 typically do not equal one another exactly (e.g.,manufacturing tolerances), but are within commercially acceptabletolerance for spacing means used in similar analysis apparatus.Spherical beads are an example of an acceptable separator 26 and arecommercially available from, for example, Bangs Laboratories of Fishers,Ind., U.S.A.

In the chamber embodiment shown in FIG. 3, the separators 26 consist ofa material that has greater flexibility than one or both of the firstpanel 12 and the second panel 16. As can be seen in FIG. 3, the largerseparators 26 are compressed to the point where most separators 26 aretouching the interior surfaces 14, 18 of the panels 12, 16, therebymaking the chamber height just slightly less than the mean separator 26diameters. In the chamber embodiment shown in FIG. 4, the separators 26consist of a material that has less flexibility than one or both of thefirst panel 12 and the second panel 16. In FIG. 4, the first panel 12 isformed from a material more flexible than the spherical separators 26and the second panel 16, and will overlay the separators 26 in atent-like fashion. In this embodiment, although small local regions ofthe chamber 10 may deviate from the desired chamber height 20, theaverage height 20 of the chamber 10 will be very close to that of themean separator 26 diameter. Analysis indicates that the mean chamberheight 20 can be controlled to one percent (1%) or better at chamberheights of less than four microns using this embodiment. Subject to theflexibility characteristics described above (as well as other factorssuch as the distribution density of the separators), the separators 26and panels 12, 16 can be made from a variety of materials, provided thepanels 12, 16 are sufficiently transparent. Transparent plastic filmsconsisting of acrylic or polystyrene are examples of acceptable panels12, 16, and spherical beads made of polystyrene, polycarbonate,silicone, and the like, are acceptable separators 26. A specific exampleof an acceptable separator is spheres made of polystyrene that arecommercially available, for example, from Thermo Scientific of Fremont,Calif., U.S.A., catalogue no. 4204A, in four micron (4 μm) diameter.Referring to FIG. 5, the panel 12 that is to be vertically disposedabove the other includes a plurality of ports 30 disposed at regularintervals (e.g., that act as air vents), and the panels 12, 16 arebonded together at points. In some embodiments, the bonding material 32forms an outer chamber wall operable to laterally contain the sample 34within the analysis chamber 10. This example of an acceptable analysischamber is described in greater detail in U.S. Patent ApplicationPublication Nos. 2007/0243117, 2007/0087442, and U.S. Provisional PatentApplication No. 61/041,783, filed Apr. 2, 2008; and 61/110,341, filedOct. 31, 2008, all of which are hereby incorporated by reference intheir entirety.

Another example of an acceptable chamber 10 is disposed in a disposablecontainer 36 as shown in FIGS. 6 and 7. The chamber 10 is formed betweena first panel 12 and a second panel 16. Both the first panel 12 and thesecond panel 16 are transparent to allow light to pass through thechamber 10. At least a portion of the first panel 12 and the secondpanel 16 are parallel with one another, and within that portion theinterior surfaces 14, 18 are separated from one another by a height 20.This chamber 10 embodiment is described in greater detail in U.S. Pat.No. 6,723,290, which patent is hereby incorporated by reference in itsentirety. The analysis chambers shown in FIGS. 2-7, represent chambersthat are acceptable for use in the present method. The present method isnot, however, limited to these particular embodiments.

An acceptable chamber height is one wherein at least some of the RBCswithin the sample individually contact both interior surfaces of thechamber panels, and/or one or more RBC aggregates contact both interiorsurfaces of the chamber panels, and one or more areas void of RBCs(e.g., lacunae) within the quiescent sample extend between the interiorsurfaces. Because the size of RBCs 22 within a blood sample is afunction of the type of blood sample being analyzed (e.g., human,monkey, horse, goat, fish, bird, etc.), the acceptable chamber heightwill vary depending on the subject being tested. A chamber height ofabout two to six microns (2-6 μm) is acceptable for individual RBCs formost animal species based on typical RBC sizes and the fact that RBCscan be deformed to some degree (e.g., the partially compressed spherediscussed above). A hematocrit analysis of an animal species having RBCssubstantially larger or smaller than human RBCs, can be performed in achamber respectively having a larger or smaller chamber height,respectively. In addition, a hematocrit analysis utilizing RBCaggregates can have a chamber height that is dictated by the height ofthe RBC aggregates.

In those chamber embodiments that do not utilize separators 26, theheight 20 of the chamber 10 can be determined as a part of themanufacturing process of the chamber and provided with the chamber.Alternatively, the height 20 of the chamber 10 can be determined using avariety of techniques including the use of a known quantity of sensiblecolorant, or the use of geometric characteristics disposed within thechamber, that can be used to determine the volume of sample for a knownfield area, and consequently the height of the chamber. These techniquesand others are described in U.S. Pat. Nos. 6,723,290 and 6,929,953. Thepresent invention is not limited to these techniques, however.

In some applications, an isovolumetric sphering agent (e.g., azwitterionic detergent or similarly functioning reagent) is admixed withat least a portion of the sample to cause at least some of the RBCs toassume a substantially spherical geometry. RBCs 22 in their natural formare often bioconcave disc shaped 38 (see FIG. 1) rather than sphericallyshaped 40. As a result, absent the effect of the isovolumetric spheringagent, a large percentage of the disc shaped RBCs 22 will not contactboth of the chamber panels 12, 16. Increasing the number of RBCs 22 thathave a substantially spherical geometry will increase the number of RBCs22 in contact with both panels 12, 16, including some cells 42 that arerestrained by the chamber panels, but would otherwise be spherical. Theisovolumetric sphering agent can be disposed in a discrete region of achamber 10 (e.g., by deposition on a particular portion of an interiorsurface). In the absence of sample mixing within the chamber 10, theagent will only admix with the portion of the sample proximate theagent, thereby leaving other sample portions untreated by the spheringagent. This selective spareing of a portion of the RBCs 22 fromisovolumetric sphering permits, as will be described below, thequalitative morphology of the RBCs 22 to be examined by image analysisas well as presentation of images to a physician for inspection forcharacteristics such as their roundness, their shape, and the presenceof protuberances on the cell. The isovolumetric sphering does notperturb any of the quantitative analyses of the RBCs 22.

The analysis of the sample quiescently disposed within the chamber 10 isperformed using an analysis device that is operable to image at least aportion of the sample and perform an analysis on the image. The image isproduced in a manner that permits the optical density of sample to bedetermined on a per unit basis. The term “per unit basis” or “imageunit” means a defined incremental unit of which the image of the samplecan be dissected. A pixelwhich is generally defined as the smallestelement of an image that can be individually processed within aparticular imaging system, is an example of an image unit, and an imageunit may also include a small number of pixels in a collective unit. Themagnification of an imaging device can also be described in linear terms(e.g., microns per pixel at the focal plane), where the linear dimensionis along a particular axis of an orthogonal grid applied to the image.The actual area of the sample captured by pixels (or other image units)of the sensor at the focal plane is therefore a function of themagnification factor applied by the imaging device. Hence, themagnification of the imaging device should be known or determinable. Thevolume associated with that pixel is therefore the area of the image perpixel times the known chamber height, since the point in the chamberthat was sensed is one where the RBC extends across the entire chamber.For example if the magnification was 0.5 microns per pixel, an imageoccupying 200 pixels would have an area of 50 square microns, and avolume of 50 square microns times the chamber height.

Now referring to FIG. 8, an example of an analysis device 44 that can beadapted for use with the present method includes a sample illuminator46, an image dissector 48, and a programmable analyzer 50. The sampleilluminator 46 includes a light source that selectively produces lightthroughout a wavelength range broad enough to be useful for thehematocrit analysis (e.g., approximately 400-670 nm; light at about 413nm and about 540 nm is particularly effective in determining the opticaldensity of the RBCs within a sample of human blood in view of the highlight absorption that occurs within the hemoglobin at the aforesaidwavelengths, which is reflected in the high molar extinction coefficient(ε) at the aforesaid wavelengths), and typically includes optics formanipulating the light. The sample illuminator 46 utilizes transmittanceto produce an image. The light transmission properties of the sample canbe measured, for example, by positioning a light source on one side ofthe sample residing within the chamber 10, directing the light throughthe sample quiescently disposed between chamber panels, and thereaftercapturing the light using the image dissector. An example of anacceptable image dissector 48 is a charge couple device (CCD) type imagesensor that converts an image of the light passing through the sampleinto an electronic data format. Complimentary metal oxide semiconductor(“CMOS”) type image sensors are another example of an image sensor thatcan be used, and the present invention is not limited to either of theseexamples. The programmable analyzer 50 includes a central processingunit (CPU) and is connected to the sample illuminator 46 and imagedissector 48. The CPU is adapted (e.g., programmed) to selectivelyperform the functions necessary to perform the present method. It shouldbe noted that the functionality of programmable analyzer 50 may beimplemented using hardware, software, firmware, or a combinationthereof. A person skilled in the art would be able to program theprocessing unit to perform the functionality described herein withoutundue experimentation. U.S. Pat. No. 6,866,823 entitled “Apparatus forAnalyzing Biologic Fluids” and issued Aug. 15, 2005, which is herebyincorporated by reference in its entirety, discloses such an analysisdevice 44.

The analysis device 44 is adapted to determine an OD value associatedwith the detected light signal on a per image unit basis for an imagedportion of the sample. The OD of a RBC 22 is determined by thehemoglobin concentration within the cell, the molar extinctioncoefficient (also referred to as molar absorptivity) for hemoglobin at agiven wavelength, and the distance of the light path traveled throughthe hemoglobin and can be represented by the following relationship:

OD=εcL

where ε=hemoglobin molar extinction coefficient, c=hemoglobinconcentration, and L=distance traveled through the RBC 22 (i.e., thehemoglobin disposed within the cell). The molar extinction coefficientis an intrinsic property of the hemoglobin that can be can be derived byexperimentation, or through empirical data currently available. Inanalysis device embodiments that utilize light sources having an errormargin (e.g., an LED having a design rated wavelength, plus or minussome amount), it is useful for accuracy purposes to initially calibratethe device and determine the hemoglobin molar extinction coefficient,which coefficient can then be used with that particular device until thelight source is replaced, at which time the device can be recalibrated.

The detected light signal (i.e., the OD values) can be used by an edgefinding algorithm to identify the locations and boundaries of RBCs. RBCs22 that contact both interior surfaces of the chamber 10 have an ODprofile similar to that of a partially compressed sphere. The lateraledges of the cell 22 that are not in contact with the surfaces 14, 18will have an OD that (in relative terms) can be considered to approachzero. The value of the determined OD: 1) increases traveling in adirection toward the center of the RBC 22 (e.g., as the lighttransmission path through the cell increases); 2) reaches a maximalvalue and remains substantially constant where the RBC is in contactwith the panel interior surfaces 14, 18 (i.e., when the transmissionlight path through the RBC is constant); and 3) decreases traveling in adirection away from the center of the RBC 22 (e.g., as the lighttransmission path through the cell decreases). This characterization ofthe OD profile of a RBC is particularly uniform for RBCs that arespherically shaped, and is not limited to those RBCs in contact withboth interior surfaces.

In some embodiments, the analysis device 44 is further adapted todetermine a mean maximal OD value for a group of RBCs 22 and/or RBCaggregates 23 in contact with both interior surfaces. The determinationof what constitutes an acceptable group size of RBCs and/or RBCaggregates in contact with the interior surfaces may be done on a persample analysis basis, or it may be done periodically for “n” number ofsample analyses of the same type; e.g., human blood samples. Forexample, a group of RBCs 22 identified as being in contact with the bothinterior surfaces 14, 18 can be comparatively evaluated to determine themean maximal OD and the statistical deviation of the OD within thegroup. It is desirable to determine the mean maximal OD because the ODof hemoglobin within the cells 22 can vary from cell to cell even withina particular sample. If the standard deviation is greater than apredetermined threshold, a new group of RBCs 22 in contact with bothpanels 12, 16 can be selected, or the existing group can be expanded,until the aforesaid analysis establishes a group of RBCs 22 having amean maximal OD with an acceptable standard deviation there from. A meanmaximal OD of the RBCs 22 within a group that is about plus or minus onepercent (1%) of the mean maximal OD of all the RBCs that contact bothsurfaces 14, 18 within the sample would, for example, be withinacceptable standard deviation values. What constitutes an acceptablestandard deviation value can, however, vary depending upon theapplication at hand and upon the specific statistical analysis beingused (e.g., standard error, etc.). Existing statistical data relating tothe OD of RBCs 22 is available and can be used in the determination ofacceptable OD statistical values. The determination of whether the RBCswithin a particular group have a mean maximal OD that is within aclinically acceptable standard deviation can also be adaptive since, asindicated above, it is well known that the population of RBCs within anindividual typically have small variations in concentration ofhemoglobin and a running standard deviation of the result can be used todetermine how many cells must be examined before a mean of acceptableaccuracy is obtained; e.g., for samples from a subject having normalblood characteristics, an acceptable group size can be as few as 100RBCs, whereas samples from a subject having abnormal bloodcharacteristics may require the analysis of 1000 or more RBCs. Thespecific number of RBCs 22 and/or RBC aggregates 23 in contact with bothinterior surfaces that are used to establish an acceptable mean maximalOD is not limited to any particular number or percentage of the RBCs 22and/or RBC aggregates 23 within a sample, and may include all (e.g.,thousands) of the RBCs 22 and/or RBC aggregates 23 in contact with bothsurfaces 14, 18.

Now referring to FIGS. 9 and 10, the analysis device 44 is furtheradapted to determine the hemoglobin concentration (“CHC”) of an RBC 22by examining a portion 25 of a RBC 22 in contact with both interiorsurfaces 14, 18 of the chamber 10. The concentration of hemoglobin isuniform within any given RBC 22. The OD value is determined on a perpixel (or other image unit) basis. The OD signal per pixel isrepresentative of the OD signal attributable to the height “L” of thechamber portion “aligned” with that pixel. In the determination of theCHC, the OD is sensed, the height of the chamber is either known or isdeterminable, and the Hemoglobin molar extinction coefficient (ε) isknown. Therefore, the CHC is determined using the relationship betweenoptical density (OD), the hemoglobin extinction coefficient (ε), and thelength of the path through the hemoglobin (L), which for a portion 25 ofa RBC 22 in contact with the interior surfaces of the chamber is equalto the height of the chamber:

OD=εcL, c=OD/εL.

The mean cell hemoglobin concentration (“MCHC”) of an RBC 22 isdetermined using the same methodology described above for determiningthe CHC of an individual RBC, repeated for some number of RBCs incontact with both interior surfaces of the chamber, using the results todetermine a mean and an acceptable standard deviation.

Now referring to FIGS. 9 and 11, the analysis device 44 is furtheradapted to determine the cell volume (“CV”) of an individual RBC 22 incontact with both interior surfaces of the chamber 10 by integrating thevolume of the RBC as a function of the OD of the hemoglobin within theRBC. The integration of the volume can be performed using a variety ofanalytical techniques. For example, according to a first technique, thecell volume of an individual RBC 22 in contact with both interiorsurfaces 14, 18 can be determined using the chamber height 20, the meanmaximal optical density determined for the portion 25 of the imaged RBC22 contacting both interior surfaces 14, 18, and the optical densitydetermined for the entire imaged RBC 22. The total optical density ofthe imaged RBC is divided by the mean maximal optical density, and theresult is corrected for the chamber height. According to anothertechnique, the cell volume is determined by dividing the individual RBC22 into different portions: the portion 25 that contacts both surfaces(“Region I”), and a portion 27 that does not contact both or even one ofthe interior surfaces 14, 18 (“Region II”). The volume of the cellportion 25 in contact with the interior surfaces 14, 18 is determined bysensing the OD of that portion 25 (i.e., Region I). The OD is sensed andis defined on a per pixel (or other image unit) basis. The chamber arearepresented by the pixel is determined, as stated above, by the size ofthe image per pixel which is a function of the magnification factor ofthe instrument. The volume associated with that pixel is therefore thearea of the image per pixel times the known chamber height, since thepoint in the chamber that was sensed is one where the RBC 22 extendsacross the entire chamber height 20. The volume of the RBC portion 25 intouch with both surfaces 14, 18 (i.e., Region I) is determined bysumming the volumes associated with each pixel within the two-surfacecontact area. The volume of the portion 27 of the RBC 22 not in contactwith both surfaces 14, 18 (i.e., Region II) is determined in a similarmanner. The OD value determined for the two-surface contact area iscompared against the OD value for each pixel within the portion 27 ofthe RBC 22 not in contact with both surfaces 14, 18 (i.e., Region II).Since the hemoglobin molar extinction coefficient (ε) is a linearfunction, the relative OD value of each pixel within Region II alsorepresents the height of the RBC 22 associated with that pixel; e.g., ifthe OD for that pixel is 50% of the OD in Region I, the height of theRBC 22 at that point is 50% of the height of the RBC in Region I (i.e.,the chamber height 20). The volume associated with each pixel in RegionII is determined on a per pixel basis as described above and summed todetermine the volume in Region II of the RBC 22. The volume of the RBC22 is the sum of Regions I and II. Decreasing the area of the image oneach pixel (i.e., increasing the resolution), increases the accuracy ofthe cell volume determination. These techniques are provided as examplesof operable techniques, and the present method is not limited to thesetechniques.

The aforesaid techniques for determining the cell volume are operable todetermine the volume of the particular RBC 22 sensed for OD. Because theOD of hemoglobin within RBCs 22 can vary from RBC to RBC even within aparticular sample, determining the volume of the cell 22 using the ODsensed for that particular cell increases the accuracy of the volumedetermination. Many RBCs 22 do not, however, contact both interiorsurfaces 14, 18 of the chamber 10. For those RBCs 22 not contacting bothsurfaces, the cell volume can be determined using the previouslyobtained mean maximal optical density of the RBCs 22 that are in contactwith both surfaces 14, 18. The mean maximal optical density obtained asdescribed is sufficiently accurate to provide an accurate volume ofthose other cells 22. As a further alternative, for those embodiments ofthe present invention that utilize an isovolumetric sphering agent, thecell volume for a RBC fragment or an RBC 22 that does not contact bothinterior surfaces 14, 18 can also be determined by assuming theaforesaid RBC fragment or RBC is spherical. If the perimeter of the RBC22 can be determined using a profiling technique as described above,that circular area can be used to determine the size of the sphere andtherefore the volume of the RBC 22.

The analysis device 44 is further adapted to determine the mean cellularvolume (“MCV”) for RBCs 22 within the sample using the same methodologydescribed above, repeated for some number of RBCs 22, using the resultsto determine a mean and a measure of the accuracy or confidence of themean; e.g., an acceptable standard error of the mean. The number of RBCs22 needed to determine a MCV with an acceptable measure of accuracy willdepend on the RBC population analyzed, which number can range from abouta few hundred to several thousand RBCs 22. One way of determiningwhether the number of RBCs whose cell volumes have been determined is anacceptable population for purposes of determining a MCV, is toiteratively determine sample mean cell volumes within the population anddetermine the standard error for those means; i.e., the standarddeviation of the means. Once the measure of accuracy (e.g., the standarderror) is within a predefined acceptable range, then the MCV isaccepted.

Now referring to FIGS. 9 and 12, the analysis device 44 is furtheradapted to determine the cell hemoglobin content (“CH”) of an RBC 22 byintegrating the hemoglobin concentration over the determined volumeoccupied by the individual RBC 22. For those RBCs 22 in contact withboth interior surfaces 14, 18 of the chamber 10, the CH is determinedusing the determined concentration (CHC) and volume (CV) for thatparticular RBC 22, as is described above. If the CH is determined on anindividual RBC 22 basis, then it can be done using the CHC determinedfor that particular RBC 22 as opposed to a mean CHC (MCHC), therebyleading to a greater degree of accuracy. For those RBCs 22 not incontact with both interior surfaces 14, 18 of the chamber 10, the CH isdetermined using the mean maximal optical density to determine theconcentration and volume for that particular RBC 22.

The analysis device 44 is further adapted to determine the mean cellhemoglobin content (“MCH”) for RBCs 22 within the sample using the samemethodology described above, repeated for some number of RBCs 22, usingthe results to determine a mean and an acceptable measure of accuracy(e.g., standard error of the mean). The number of RBCs 22 needed todetermine an MCH with an acceptable measure of accuracy will depend onthe RBC population analyzed, which number can range from about a fewhundred to several thousand RBCs 22. Once the measure of accuracy (e.g.,the standard error) is within an acceptable range, then the MCH isaccepted.

The above described methodologies for determining the CHC, MCHC, CV,MCV, CH, and MCH are examples of how these characteristics of asubstantially undiluted blood sample can be determined using thedescribed chamber and analytical device with the present invention. Thepresent invention is not limited to these specific examples.

Under the present method, a sample of substantially undiluted wholeblood is placed in a chamber 10 as is described above. Ananticoagulating agent, and in some instances an isovolumetric spheringagent and/or an aggregating agent, is mixed with the sample either priorto its introduction into the chamber or upon introduction into thechamber. Reagents added in dry or semi-dry form, for example via surfacecoating, are particularly easy to use. The present invention is notlimited to dry form reagents, however, and can for example use liquidreagents that do not meaningfully dilute the sample. The samplequiescently resides within the chamber. Under certain circumstances(e.g., very rapid analysis), it may not be necessary to add theanticoagulating agent, but it is preferable to do so in most cases toensure the sample is in a form acceptable for analysis. In certainanalyses (e.g., those that provide information on individual RBCs), itmay be preferable to not include an aggregating reagent.

At least a portion of the sample quiescently residing within the chamber10 is imaged using the analysis device 44 by transmitting light throughthe sample and detecting the transmitted light. Although it is not arequirement that the entire sample residing within the chamber 10 beimaged, it is preferable since doing so typically provides a morecomplete analysis of the sample (and all of its constituents) and aconcomitant increase in accuracy since the distribution of RBCs 22 andRBC void areas 24 within a chamber is typically non-homogeneous for asample of substantially undiluted whole blood.

A group of RBCs 22 in contact with the interior surfaces 12, 16 of thechamber 10 is determined by the analysis device 44 using the image ofthe sample portion. Depending upon which characteristic of the bloodsample is requested, the analysis device 44 will determine one or moreof the characteristic values to arrive at the requested characteristicvalue.

An advantage of the present invention is that it is not necessary tohave all of the RBCs 22 within the sample contact each chamber panel.The method can be performed with only some of the RBCs 22 in contactwith both interior surfaces 14, 18 of the chamber 10. Smaller RBCs 22and RBC fragments are not used to calibrate the analysis, but aremeasured for their contribution to the hematocrit. In addition, underthe present invention the CHC, MCHC, CV, MCV, CH, and MCH of the samplecan be determined without knowledge of the total area or volume of thesample within the chamber 10.

The RBCs 22 identified and analyzed under the present invention includereticulocytes (immature red blood cells), which cells develop in bonemarrow as nucleated cells. Before the reticulocytes are released intocirculation, they are stripped of their nuclei. The reticulocytescirculate for about a day in the blood stream before losing theirreticular staining (which is a function of stained remnants ofcytoplasmic RNA and nuclear DNA) and develop into mature RBCs 22containing essentially only hemoglobin. The relative number ofreticulocytes within the blood sample can be an important indicator ofvarious conditions. For example, the number of reticulocytes is a goodindicator of bone marrow red cell production activity, because itrepresents recent production. Abnormally absolute low numbers ofreticulocytes can be indicative of aplastic anemia, pernicious anemia,bone marrow malignancies, problems of erythropoietin production, variousvitamin or mineral deficiencies (B9, B12, and iron), etc. Abnormallyhigh absolute reticulocyte counts may indicate rapid production due tothe body's replacement of blood loss due to hemorrhage or hemolysis.Consequently, there is significant value in being able to detect andenumerate reticulocytes. Under the present invention, reticulocytes areidentified as a RBC 22 because they contain hemoglobin, and because theycan be stained to identify remaining RNA and DNA.

The reticulocytes can also be distinguished from other RBCs 22 andenumerated by admixing the sample with a stain such as a supravital dyesuch as acridine orange, astrozone orange, or the like. The dye causesthe reticulin naturally present within the reticulocytes to fluorescewhen excited by violet light at about 470nm. The location of the RBCs 22within the quiescent sample can be determined via the RBCs' opticaldensity due to the contained hemoglobin in all reticulocytes anddetermined from the image of the sample. The reticulocytes can bedistinguished from RBCs 22 not containing reticulin and from white bloodcells by imaging and examining under fluorescence the sample at one ormore selected wavelengths (e.g., 470 nm) associated with the supravitaldye. For those RBCs identified as reticulocytes by the presence ofhemoglobin and the florescence of the reticulin, the above describedmethodologies for determining the cell volume, cell hemoglobin content,and cell hemoglobin concentration can be used to determine the same forindividual reticulocytes. In addition, statistical information (e.g.,mean values and measures of accuracy) can be determined. The relativequantity of reticulin within each reticulocyte, which varies inverselywith the maturity of the reticulocyte, can also be determined by theintensity of the fluorescent signal. As a result, even more specificinformation can be determined regarding individual reticulocytes as wellas the population of reticulocytes. The relative amount of reticulin inan individual reticulocyte can be determined using either the area offluorescence or the intensity of the fluorescence, and may be calculatedas a function of the volume of an individual reticulocyte.

Using the present invention, RBC indices can also be determinedincluding or excluding reticulocytes. For example, the MCV of a samplemay be plotted both with and without including the volume contributiondue to reticulocytes. Determining the MCV without reticulocytes canunmask population microcytosis that is affected by the large size ofreticulocytes. Multiple other mathematical relationships among the RBCsand the reticulocytes may be determined.

As indicated above, individual indices can be determined under thepresent invention for RBCs 22 that do not contact both panel interiorsurfaces 14, 18. RBC fragments can be analyzed in the same manner,thereby permitting RBC fragments to be distinguished from otherconstituents found within a blood sample; e.g., platelets, plateletclumps, white cell fragments, debris, etc. The ability to detect RBCfragments using the present invention is particularly useful because RBCfragments can be indicative of conditions such as microangiopathicanemia, severe inflammation, and disseminated malignancy. The analysisof RBC fragments within a blood sample that has not been treated with anisovolumetric sphering agent can, for example, include morphologicalanalyses to determine unaltered morphology characteristics such as sizedeterminations, deviation from roundness, perimeter to area ratio, andthe like. The OD image produced using the present invention permits thedetermination of characteristics such as sharpness, ellipticity,protrusions, etc., for each RBC fragment, which characteristicsfacilitate the aforesaid morphological analyses. Additionally, thevolume of red cell fragments may be determined by measuring theirdiameter or circumference and calculating the volume of a sphere withthose dimensions.

Although this invention has been shown and described with respect to thedetailed embodiments thereof, it will be understood by those skilled inthe art that various changes in form and detail may be made withoutdeparting from the spirit and scope of the invention.

1. A method for determining red blood cell hemoglobin concentrationwithin a blood sample, comprising the steps of: depositing the sampleinto an analysis chamber adapted to quiescently hold the sample foranalysis, the chamber defined by an interior surface of a first panel,and an interior surface of a second panel, wherein both panels aretransparent, and the chamber has a known or determinable heightextending between the interior surfaces of the panels, which height issuch that at least one red blood cell within the sample contacts both ofthe interior surfaces; imaging at least one red blood cell contactingthe interior surfaces; determining an optical density of at least aportion of the imaged red blood cell contacting both interior surfaces;and determining the hemoglobin concentration of the red blood cellcontacting the interior surfaces, using the determined optical density.2. The method of claim 1, wherein the optical density is determined on aper image unit basis
 3. The method of claim 2, wherein the image unit isa pixel.
 4. The method of claim 1, further comprising the steps of:determining the hemoglobin concentration of a plurality of red bloodcells contacting the interior surfaces; and determining a meanhemoglobin concentration using the hemoglobin concentrations determinedfor each of the plurality of red blood cells.
 5. The method of claim 4,wherein the entire sample is imaged.
 6. The method of claim 1, furthercomprising the step of admixing an isovolumetric sphering agent with atleast a portion of the sample.
 7. The method of claim 6, wherein theisovolumetric sphering agent is a zwitterionic detergent.
 8. The methodof claim 7, wherein the interior surfaces of the panels aresubstantially parallel, and the chamber height is known prior to thedetermination of the hemoglobin concentration.
 9. The method of claim 8,wherein the chamber height is within the range of about two microns tosix microns.
 10. The method of claim 1, further comprising the steps of:imaging a plurality of red blood cells, at least a portion of each suchred blood cell contacting the interior surfaces; determining an opticaldensity of the portion of each such red blood cell contacting bothinterior surfaces; determining a mean maximal optical density using theoptical densities determined for at least some of the plurality of redblood cells; and determining the hemoglobin concentration of one or morered blood cells within the sample not in contact with both interiorsurfaces, using the determined mean maximal optical density.
 11. Themethod of claim 1, wherein the sample has a first portion admixed withan isovolumetric sphering agent and a second portion free of theisovolumetric sphering agent.
 12. The method of claim 11, wherein atleast some of the one or more red blood cells not in contact with bothinterior surfaces reside within the second portion of the sample, andfurther comprising the step of determining an unaltered morphology ofsome of the red blood cells within the second portion that are not incontact with both interior surfaces.
 13. The method of claim 1, furthercomprising the step of admixing a supravital dye with the sample, whichdye is operable to cause reticulin within reticulocytes within thesample to fluoresce when excited by light of one or more predeterminedwavelengths.
 14. The method of claim 13, further comprising the step ofdetermining a relative quantity of reticulin within one or morefluorescing reticulocytes either as the area of fluorescence or theintensity of fluorescence.
 15. The method of claim 13, furthercomprising the steps of: imaging a plurality of red blood cells, atleast a portion of each such red blood cell contacting the interiorsurfaces, wherein the imaging is performed using light at one or morepredetermined wavelengths that is absorbed by hemoglobin disposed withinthe red blood cells; imaging the plurality of red blood cells at thepredetermined wavelengths operable to cause the reticulin within thereticulocytes to fluoresce; determining an optical density of theportion of each such red blood cell contacting both interior surfaces;determining the hemoglobin concentration of each of the plurality of redblood cells, excluding those red blood cells that fluoresce, using thedetermined optical density value; and determining a mean hemoglobinconcentration using the hemoglobin concentrations determined for each ofthe plurality of red blood cells, excluding those red blood cells thatfluoresce.
 16. The method of claim 1, wherein the step of imaging isperformed at one or more predetermined wavelengths, and thedetermination of the hemoglobin concentration utilizes a molarextinction coefficient of hemoglobin for the one or more predeterminedwavelengths.
 17. The method of claim 16, wherein the step of imaging isperformed using an analytical device calibrated to determine acalibrated hemoglobin molar extinction coefficient value for thatanalytical device.
 18. The method of claim 1, wherein the blood sampleis substantially undiluted.
 19. The method of claim 1, wherein the bloodsample is whole blood.
 20. A method for determining a cell volume of ared blood cell within a blood sample, comprising the steps of:depositing the sample into an analysis chamber adapted to quiescentlyhold the sample for analysis, the chamber defined by an interior surfaceof a first panel, and an interior surface of a second panel, whereinboth panels are transparent, and the chamber has a known or determinableheight extending between the interior surfaces of the panels, whichheight is such that at least one red blood cell within the samplecontacts both of the interior surfaces; imaging the at least one redblood cell contacting the interior surfaces, and producing imagesignals; determining a mean optical density of a portion of the redblood cell in contact with both interior surfaces, and a total opticaldensity of the red blood cell, both on a per image unit basis; anddetermining a volume of the imaged red blood cell using the mean opticaldensity of the portion of the cell in contact with the interiorsurfaces, the total optical density, and the height of the chamber. 21.The method of claim 20, wherein the blood sample is substantiallyundiluted.
 22. The method of claim 20 wherein the blood sample is wholeblood.
 23. The method of claim 20, wherein the image unit is a pixel.24. A method for determining a cell volume of a red blood cell within ablood sample, comprising the steps of: depositing the blood sample intoan analysis chamber adapted to quiescently hold the sample for analysis,the chamber defined by an interior surface of a first panel, and aninterior surface of a second panel, wherein both panels are transparent,and the chamber has a known or determinable height extending between theinterior surfaces of the panels, which height is such that at least onered blood cell within the sample contacts both of the interior surfaces;imaging the at least one red blood cell contacting the interiorsurfaces, including a portion of the red blood cell in contact with bothinterior surfaces and a portion not in contact with both interiorsurfaces; determining an optical density of the portion of the red bloodcell in contact with both interior surfaces, and an optical density ofthe portion not in contact with both interior surfaces on a per imageunit basis; determining a volume of the portion of the imaged red bloodcell contacting both interior surfaces using the per image unit opticaldensity determined for that portion; determining a volume of the portionof the imaged red blood cell not in contact with both interior surfacesusing the per image unit optical density determined for that portion,and the per image unit optical density determined for the portion incontact with both surfaces; and determining the cell volume of the atleast one red blood cell using the volume determined for the portion ofthe imaged red blood cell contacting both interior surfaces and thevolume determined for the portion of the imaged red blood cell not incontact with both interior surfaces.
 25. The method of claim 24,comprising the steps of: determining the cell volume for a plurality ofred blood cells in contact with both interior surfaces; and determininga mean cell volume using the cell volume determined for each of theplurality of red blood cells.
 26. The method of claim 24, wherein theentire sample is imaged.
 27. The method of claim 24, further comprisingthe step of admixing an isovolumetric sphering agent with at least aportion of the sample.
 28. The method of claim 27, wherein theisovolumetric sphering agent is a zwitterionic detergent.
 29. The methodof claim 24, wherein the interior surfaces of the panels aresubstantially parallel, and the chamber height is known prior to thedetermination of the hemoglobin concentration.
 30. The method of claim29, wherein the chamber height is within the range of about two micronsto six microns.
 31. The method of claim 24, further comprising the stepsof: imaging a plurality of red blood cells within the sample, eachhaving a portion in contact with both interior surfaces; determining foreach red blood cell, an optical density of the portion of that red bloodcell in contact with both interior surfaces on a per image unit basis;and determining a mean maximal optical density using the optical densitydetermined for the portion of each of the plurality of red blood cellsin contact with both interior surfaces.
 32. The method of claim 31,further comprising the steps of: imaging one or more red blood cells notin contact with both interior surfaces; determining an optical densityof the red blood cells not in contact with both interior surfaces;determining a cell volume for the one or more red blood cells not incontact with both interior surfaces, using the optical density of thatred blood cell, the determined mean maximal optical density, and theheight of the chamber.
 33. The method of claim 32, wherein the samplehas a first portion admixed with an isovolumetric sphering agent and asecond portion free of the isovolumetric sphering agent.
 34. The methodof claim 33, wherein at least some of the one or more red blood cellsnot in contact with both interior surfaces reside within the secondportion of the sample, and further comprising the step of determiningthe unaltered morphology at least one of the one or more red blood cellswithin the second portion that are not in contact with both interiorsurfaces.
 35. The method of claim 31, further comprising the steps of:imaging at least one red blood cell not in contact with both interiorsurfaces; determining the optical density of the red blood cell not incontact with both interior surfaces; determining the hemoglobinconcentration of the red blood cell not in contact with both interiorsurfaces, using the mean optical density determined for the red bloodcells in contact with both interior surfaces.
 36. The method of claim24, further comprising the step of admixing a supravital dye with thesample, which dye is operable to cause reticulin within reticulocyteswithin the sample to fluoresce when excited by light of one or morepredetermined wavelengths.
 37. The method of claim 36, furthercomprising the step of determining a relative quantity of reticulinwithin one or more fluorescing reticulocytes.
 38. The method of claim36, further comprising the steps of: imaging a plurality of red bloodcells, at least a portion of each such red blood cell contacting theinterior surfaces, wherein the imaging is performed using light that isabsorbed by hemoglobin disposed within the red blood cells; imaging theplurality of red blood cells at the predetermined wavelengths operableto cause the reticulin disposed within the reticulocytes within thesample to fluoresce; determining the cell volume for each of theplurality of red blood cells; and determining a mean cell volume usingthe cell volume for each of the plurality of red blood cells, excludingfrom the mean cell volume the cell volume of each red blood cell thatfluoresces.
 39. The method of claim 24, wherein the step of imaging isperformed at one or more predetermined wavelengths, and thedetermination of the hemoglobin concentration utilizes a molarextinction coefficient of hemoglobin for the one or more predeterminedwavelengths.
 40. The method of claim 24, further comprising the stepsof: determining the hemoglobin concentration of the red blood cell,using the optical density determined for the portion of the red bloodcell in contact with both interior surfaces; and determining thehemoglobin content within the red blood cell using the hemoglobinconcentration and the cell volume.
 41. The method of claim 40, furthercomprising the steps of: determining the hemoglobin content within aplurality of red blood cells, each having a portion in contact with bothinterior surfaces; determining a mean hemoglobin content using thehemoglobin content determined for each of the plurality of red bloodcells.
 42. The method of claim 24, wherein the blood sample issubstantially undiluted.
 43. The method of claim 24, wherein the bloodsample is whole blood.
 44. The method of claim 24, wherein the imageunit is a pixel.
 45. An apparatus for determining red blood cellhemoglobin concentration within a substantially undiluted blood samplehaving a plurality of red blood cells, comprising: an analysis chamberadapted to quiescently hold the sample for analysis, the chamber definedby an interior surface of a first panel, and an interior surface of asecond panel, wherein both panels are transparent, and the chamber has aknown or determinable height extending between the interior surfaces ofthe panels, which height is such that one or more of the red blood cellswithin the sample will contact both of the interior surfaces; an imagingunit that includes an illuminator and an image dissector, which unit isoperable to image one or more red blood cells contacting the interiorsurfaces, and produce image signals representative of such imaged redblood cells; and a programmable analyzer adapted to determine, using theimage signals, an optical density of at least a portion of the imagedred blood cells contacting both interior surfaces on a per image unitbasis, and the hemoglobin concentration of the red blood cellscontacting the interior surfaces, using the determined optical density.46. An apparatus for determining a cell volume of at least one red bloodcell within a substantially undiluted blood sample, comprising: ananalysis chamber adapted to quiescently hold the sample for analysis,the chamber defined by an interior surface of a first panel, and aninterior surface of a second panel, wherein both panels are transparent,and the chamber has a known or determinable height extending between theinterior surfaces of the panels, which height is such that the at leastone red blood cell contacts both of the interior surfaces; an imagingunit that includes an illuminator and an image dissector, which unit isoperable to image the at least one red blood cell contacting theinterior surfaces, and produce image signals representative of suchimaged red blood cell; and a programmable analyzer adapted to determine,using the image signals, a mean optical density of the red blood cellscontacting both interior surfaces and a total optical density of the redblood cell, and a volume of the imaged red blood cell using the meanoptical density, the total optical density, and the height of thechamber.